Field of the Invention
The present relates to a method for enhancing cellobiose utilization ability of yeast, and in particular, for enhancing intracellular cellobiose utilization of Saccharomyces cerevisiae. 
Background Art
The potential of plant biomass as a cheap and renewable substrate for the production of fuel and chemicals has gained considerable interest in recent years. The biological saccharification of cellulose, the main component of plant biomass, is of particular interest in the field of fuel ethanol production. Four, biologically mediated process steps are involved in the current cellulose-to-ethanol technology: (i) cellulase enzyme production; (ii) enzymatic saccharification of cellulose; (iii) fermentation of hexose sugars (end-products of cellulose hydrolysis); and (iv) fermentation of pentose sugars (end-products of hemicellulose hydrolysis) to ethanol (Lynd, L. R., et al., Microbiol. Mol. Biol. Rev. 66:506-577 (2002)). Combining all four process steps into a one-step conversion of cellulose to fuel ethanol (called consolidated bioprocessing (CBP)) would result in a considerable reduction in processing costs (Lynd, L. R., et al., Microbiol. Mol. Biol. Rev. 66:506-577 (2002)).
Saccharomyces cerevisiae has superior ethanol formation properties, but is non-cellulolytic. The expression of cellulases in S. cerevisiae would be a prerequisite for cellulose conversion via CBP. S. cerevisiae has received a great deal of interest regarding heterologous protein expression as well as the production of ethanol and other commodity product (Lynd, L. R., et al., Microbiol. Mol. Biol. Rev. 66:506-577 (2002)); (Romanos, M. S, et al., Yeast 8:423-88 (1992)). Expression of a functional cellulase system in S. cerevisiae would require the co-expression of at least three groups of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21). These enzymes act synergistically to efficiently degrade cellulose (Mansfield and Meder; 2003). β-Glucosidases catalyze the hydrolysis of soluble cellodextrins and cellobiose to glucose. β-glucosidases from various origins, e.g. Aspergillus niger (Dan, S., et al. J Biol Chem 275:4973-4980 (2000)), Aspergillus kawachii (Van Rooyen, R., et al., J. Biotechnol. 120:284-295 (2005); Iwashita, K. T. Nagahara, et al., Appl Environ Microbiol 65:5546-5553 (1999)) Candida pelliculosa var. acetaetherius (Kohchi C. and A. Toh-e, Mol Gen Genet 203:89-94 (1986)), Candida wickerhamii (Van Rooyen, R., et al., J Biotechnol. 120:284-295 (2005)), Saccharomycopsis fibuligera and Trichoderma reesei (Van Rooyen, R., et al., J. Biotechnol. 120:284-295 (2005)) have been successfully expressed in S. cerevisiae. This previous work focused on secreted β-glucosidases. Raynal A. and M. Guérineau, et al., Mol Gen Genet 195:108-115 (1984) have genetically engineered S. cerevisiae to produce the Kluyveromyces lactis β-glucosidase intracellularly, but the recombinant strain was unable to grow on cellobiose.
Previous work of the applicant describes the construction of cellobiose-fermenting strains of S. cerevisiae by introduction of secreted β-glucosidases from various fungal origins (Van Rooyen, R., et al., J. Biotechnol. 120:284-295 (2005)). The accumulation of extracellular cellobiose has two major disadvantages: (i) it causes feedback inhibition of endoglucanases and cellobiohydrolases and therefore limits the rate and extent of cellulose hydrolysis (Yan, T., et al., J. Agric. Food. Chem. 46:431-437 (1998)); and (ii) the action of β-glucosidases releases glucose in the external environment that increases the risk of contamination.
There is therefore a need for a method for enhancing intracellular cellobiose utilization by a host cell such as S. cerevisiae, which does not have the problems described above.